Water soluble nanocrystalline quantum dots

ABSTRACT

An economic, direct synthetic method for producing water soluble QDs that are ready for bioconjugation is provided. The method can produce aqueous QDs with emission wavelengths varying from 400 nm to 700 nm. Highly luminescent metal sulfide (MS) QDs are produced via an aqueous synthesis route. MS QDs are capped with thiol-containing charged molecules in a single step. The resultant MS QDs exhibit the distinctive excitonic photoluminescence desired of QDs and can be fabricated to avoid undesirable broadband emissions at higher wavelengths. This provides a significant improvement over the present complex and expensive commercial processes for the production of QDs. The aqueous QDs are stable in biological fluids over a long period of time. In addition, nontoxic ZnS QDs have been produced with good photoluminescence properties by refluxing the ZnS QD suspensions over a period of time.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. patent application Ser. No.12/887,224, filed on Sep. 21, 2010, currently pending, which, in turn,is a continuation of U.S. patent application Ser. No. 12/552,970, filedon Sep. 2, 2009, now U.S. Pat. No. 7,824,653; which, in turn, is acontinuation of U.S. patent application Ser. No. 11/968,228, filed onJan. 2, 2008, now U.S. Pat. No. 7,597,870; which, in turn, is acontinuation of U.S. patent application Ser. No. 11/136,653, filed onMay 24, 2005, now U.S. Pat. No. 7,335,345; which, in turn, is anonprovisional of U.S. provisional patent application No. 60/573,804,filed on May 24, 2004, the disclosures of entireties of which areincorporated by reference herein in their entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was reduced to practice with Government support under R01EB00720-01 awarded by NIH; the government is therefore entitled tocertain rights to this invention.

BACKGROUND OF THE INVENTION

1. Field of Invention

The present invention relates to the aqueous synthesis ofphotoluminescent nanocrystalline quantum dots.

2. Brief Description of the Prior Art

Semiconductor nanocrystalline quantum dots (QDs) with bioconjugates onthe surface have been studied extensively because of their uniqueoptical properties. QDs are inorganic nanoparticles that emit light at aspecific wavelength when excited by light. When light shines on QDs,electrons in the valence band are excited to the conduction band,forming short-lived (nanoseconds) electron-hole pairs called excitons,which emit photons of a specific wavelength when the electron-hole pairseventually recombine. The excitonic emission is independent of thewavelength of the excitation light. This makes it easier to excite QDsto luminescence than the traditional fluorescent molecules that requirea specific excitation wavelength. The wavelength of the emitted photonsof QDs, on the other hand, is specific and can be controlled by the QDs'particle size and composition. The synthesis of QDs was developed mostlyin the 1990's. In the last few years, the interest in using QDs inbiomedical imaging has exploded due to advances in surface modificationof QDs that have made them accessible for antibody immobilization anddetection of antibody-antigen binding.

Using QDs as imaging markers inside living organisms is one of theexciting new nanobiotechnologies. QDs can be used as biological markersto find a disease as well as to carry a drug to the exact cell thatneeds it by immobilizing antibodies on the surface of the QDs. QDs maybe specific to a particular disease and may be tailored to bind only toinfected cells. Detection may be carried out either by locating the QDs'particles or by detecting signals emanating from the QDs' particles. Forexample, luminescence of antibody-coated QDs bound to the canceroustissue in a mouse helped locate the tumor.¹ Until now the mainbiological tags that have been employed are organic fluorophores orradioactive labels.² Radioactive labels are short lived and radioactive.Concerns about the use of radioactive materials in the body alwaysarise. Organic fluorophores have wide emission spectra and the emissionis not as bright as that of QDs. In comparison to conventional dyemolecules, QDs have the advantages of having tunable fluorescencesignatures, narrow emission spectra, brighter emissions, and goodphotostability.³ Due to the enormous interest in using QDs as biologicaltags, QDs are now commercially available from quite a number ofcompanies. However, the complexity of the existing organic-basedsynthesis route for fabricating commercial QDs makes the priceprohibitively high, as much as U.S. $1200/g without bio-conjugation⁴,and $3200/mg for bioconjugated QDs.⁵ Part of the complexity of theexisting QDs production technology stems from the need to improve thephotoluminescence yield by eliminating the broadband emission of earlierQDs by capping with an inorganic layer. Making QDs water-soluble isanother challenge for biomedical applications.

Both groups II-VI nanocrystals such as CdSe, CdTe, CdS,^(6,7) ZnS,⁸ andZnSe, and groups III-V nanocrystals such as InP and InAs have beensynthesized and studied extensively in the past.⁹ One type of quantumdot currently on the market is based on CdSe nanocrystals capped by, forexample, ZnS. The synthesis follows the method popularized by Bawendi'sgroup at MIT involving the pyrolysis of organometallic precursors,dimethylcadmium and trioctylphosphine oxide (TOPO) to form CdSenanocrystals. ZnS capping on CdSe was done using diethylzinc andhexamethyldisilathiane precursors.¹⁰

Alivisatos and coworkers further made QDs water-soluble by addition of asilica/siloxane coating.¹¹ With a silica coating, 3-(mercaptopropyl)trimethoxysilane (MPS) is then adsorbed on the nanocrystals anddisplaces the TOPO molecules, making the surface of the QDs suitable forantibody immobilization.¹² These processes are complex involvingmultiple steps and a change of solvent from organic to aqueous duringthe process.

An aqueous process for the manufacture of CdS QDs was published recentlyusing adenosine triphosphate (ATP) as the capping molecule.¹³ Thisprocess suffers from the disadvantage that the luminescence spectrum ofthe resultant CdS QDs includes an undesirable non-excitonic broadbandemission between 500 nm to 700 nm wavelength.

Rogach et al.¹⁴ describes the synthesis of oxidation-stable CdTenanoclusters in aqueous solution using 2-mercaptoethanol and1-thioglycerol as stabilizers. CdTe nanocrystals generally have noluminescent properties until they are stabilized with 2-mercaptoethanol.However, this capping method also yields QDs with an undesirablebroadband emission at higher wavelengths. Similarly, when CdTe wasstabilized with thioglycerol, a broadband emission at higher wavelengthswas also observed. Currently, only the capping of inorganic materials,for example, a ZnSe shell on CdTe core, can eliminate the undesirablebroadband emission. Thus, there remains a need for an economic, directaqueous synthesis route for the production of highly luminescentwater-soluble nanocrystalline QDs.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a process for theproduction of QDs in aqueous media, which are ready for bioconjugation.In another aspect, the present invention relates to quantum dots made bythe process. These quantum dots are ready for bioconjugation and mayexhibit improved emission spectra, relative to other QDs fabricated inaqueous media.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows emissions of CdS and ZnS-doped CdS QDs excited with a UVlamp. From left to right (red to blue), the emission maxima are locatedat 660, 620, 550, 515 and 475 nm.

FIG. 2 shows integrated photoluminescence intensity vs. absorbance forRhodamine 101, EviTags™ CdSe/ZnS QDs and aqueous CdS QDs.

FIG. 3 shows normalized PL intensity when excited by UV light of awavelength of 365 nm. The intensity is normalized by the concentrationand the peak PL of the lowest concentration is set to be 1.

FIG. 4 shows the PL intensity of aqueous CdS QDs as a function of timein water, PBS, and cytosol, respectively.

FIG. 5 shows the PL intensity of aqueous CdS QDs with different amountof ZnS doping. As Zn content increases, the peak shifts to a lowerwavelength.

FIG. 6 shows the PL intensity of ZnS after reflux for different timeperiods. Reflux at 100° C. for 16 hrs gave the highest intensity in thisexample.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a first aspect, the present invention relates to an economic, directsynthetic method for producing water soluble QDs that are ready forbioconjugation. Highly luminescent metal sulfide (MS) QDs are producedvia an aqueous synthesis route. The resultant MS QDs may then be cappedwith carboxylated molecules in a single step. The capped MS QDsgenerally exhibit the distinctive excitonic photoluminescence desired ofQDs without the undesirable broadband emission at higher wavelengthsoften present in other QDs fabricated in aqueous media, indicating thatthe aqueous process of the present invention is effective in producingthe substantially clean MS surface that appears to be necessary for ahigh luminescence yield. This is significant because, as far as theinventors are aware, no known aqueous method has completely eliminatedthe tendency for the QDs to exhibit an undesirable broadband emission athigher wavelengths.

Furthermore, the carboxylated molecules used to cap the particle surfaceof the MS QDs render the MS QDs ready for surface immobilization ofantibodies and other biomolecules. That the surface of the MS QDs isimmediately ready for further biological modification in a single stepalso represents a significant advantage over the manufacture of QDs byother methods. This results in substantially reduced material costs. Inaddition to the economic benefits and the potential impact onfundamental research in the field of surface chemistry, the aqueousprocessing route of the present invention is environmentally friendlyand readily adapted to commercial production levels.

In general, the manufacturing process of the present invention may beapplied to fabricate QDs from water-soluble precursors. Thus, any QDsthat can be made from water-soluble precursors are within the scope ofthe present invention.

In the manufacturing process of the present invention, any salt of ametal suitable for use in a quantum dot, that is soluble in water, maybe employed as a starting material. Exemplary water-soluble metal saltsthat may be employed in the invention are metals that can form sulfides,such as Cd(NO₃)₂, Cd(ClO₄)₂, CdCl₂, CdSO₄, cadmium acetate, Zn(NO₃)₂,Zn(ClO₄)₂, ZnSO₄, ZnCl₂, zinc acetate, Mn(NO₃)₂, Mn(ClO₄)₂, MnSO₄,MnCl₂, manganese acetate, Pb(NO₃)₂, Pb(ClO₄)₂, PbSO₄, PbCl₂, and leadacetate.

Any suitable water-soluble sulfide may be used as a reactant in theprocess of the invention. Exemplary water-soluble sulfides that may beemployed in the invention are sulfides such as CdS, NaS, ZnS and PbS.Also, sulfide gases, such as H₂S, may be bubbled through the aqueoussolution in the process of the invention. The addition of sulfide ispreferably done gradually, such as by titration, with stiffing, and maytake, for example, about 2 hours. Generally, it is desirable to useabout a stoichiometric amount of the sulfide. However, varying theamount of sulfide from a stoichiometric amount can, in some cases,produce desirable variations in the particle sizes of the particles inthe QDs and thus, it may be useful to use anywhere from 0.1 to 10 timesthe stoichiometric amount of sulfide, more preferably 0.5 to 5 times thestoichiometric amount of the sulfide, and most preferably about 0.8-1.2times the stoichiometric amount of the sulfide. The stoichiometricamount is based on the reaction of the sulfide with the metal to formthe metal sulfide.

Also, any thiol-functionalized molecule with a charged group, preferablyon the opposite end, may be used as a reactant in the process of theinvention, as long as the thiol-functionalized molecule iswater-soluble. Exemplary thiol-functionalized molecules for use in thepresent invention include 4-aminothiophenol, mercaptosilanes such as3-mercaptopropyltrimethoxysilane, and similar materials, as well asmercaptocarboxylic acids such as mercaptoacetic acid, mercaptopropionicacid, mercaptosuccinic acid, mercaptobenzoic acid, andmercaptoundecanoic acid. Any concentration of thiol-functionalizedmolecule may be employed, as long as it is within the solubility limitof the thiol-functionalized molecule in aqueous media.

The ratio of the various reactants is not critical and, in fact, may bevaried in order to customize the particle size of the resultant cappedQDs. In generally, however, the molar ratio of thiol groups to metal mayvary from about 1 to about 100, though ratios of 1-5 are more preferred,with a ratio of about 2 being most preferred.

In one embodiment of the invention, highly luminescent CdS QDs that arecapped with mercaptocarboxylic acids (MCA) in a single step, aresynthesized. Fluorescence results indicate that the CdS QDs exhibitbright fluorescence in the visible range from blue to red as exemplifiedin FIG. 1. In FIG. 1, the emissions of CdS QDs synthesized by the methodof the present invention are shown. These emissions exhibit differentfluorescent colors when excited by a 365 nm UV lamp. Any water-solubleCd salt may be used, including but not limited to, cadmium nitrate,cadmium acetate, cadmium chloride, cadmium hydroxide, and cadmiumsulfate. The molar ratios between the MCA and Cd may vary from about 1to about 100. More preferably, the molar ratio between the MCA and theCd is from about 1.5 to about 3, and most preferably about 2.

One advantage of certain embodiments of the present invention is theelimination of the broadband emission of the resultant QDs. A secondadvantage of certain embodiments of the invention is that a one-step,aqueous process produces QDs capped with COO⁻, which can readily beactivated to form a peptide bond with an amine group of a protein, i.e.,is ready for antibody/receptor immobilization on the QD surface. Atypical reaction time for the step of chelating Cd with MCA is about 12hours, though the reaction time may vary depending on the specificreactants and reaction conditions employed. It will be understood bythose of ordinary skill in the art that reaction times and conversionrates are not critical to the process of the present invention.

In a more preferred aspect, CdS QDs are synthesized using3-mercaptopropionic acid (HSCH₂CH₂COOH) (MPA), cadmium nitrate(Cd(NO₃)₂) and sodium sulfide (Na₂S). For biological applications, suchas biomarkers, where antibodies need to be conjugated to the QDs,aqueous suspensions of QDs are most desirable since the conjugationprocess can be accomplished directly in the aqueous suspension withoutfurther preparative steps. However, precipitates may be produced forshipping or other reasons. Such precipitates can be reconstituted beforeuse, if desirable.

In another aspect, a capping molecule capable of chelating with Cd ionsto minimize the formation of impurity states due to dangling Cd ions isrequired. These capping molecules should as act to stabilize and limitthe growth of the particles. 3-mercaptopropionic acid (HSCH₂CH₂COOH)(MPA) is preferred as the capping molecule because it has a thiol groupthat can bind to Cd. This follows the example of synthesizingmonodispersed gold suspensions using sodium citrate.¹⁵ Citrate not onlyreduces the gold but also serves as the capping molecule to stabilizethe gold particles. By varying the ratio of citrate to gold, goldparticle size is controlled.¹⁶ Without being bound by theory, MPA mayplay a similar role to cap and stabilize CdS particles in the QDs.

Occasionally, difficulties may arise during the reaction if the pH ofthe reaction mixture is in the vicinity of the isoelectric point (IEP)of the solution. Thus, in such cases, it may be desirable to adjust thepH of the reaction mixture away from the IEP using a suitable,water-soluble pH-adjusting agent, before the addition of sodium sulfide.One example of a suitable pH-adjusting agent is ammonium hydroxide. Theconcentration of the pH-adjusting agent may be varied, as necessary, toproduce optimum results. Preferred concentrations of ammonium hydroxideare in the range of about 0.5-2M and, more preferably, about 0.8-1.2 M,with about 1M being the most preferred concentration of the ammoniumhydroxide pH-adjusting agent.

After adjustment of the pH away from the IEP of the metal sulfide,sodium sulfide is added quickly to minimize the reaction time. A fewminutes of reaction time is sufficient. The process is best performed inoxygen-free environment to avoid the photo-oxidation reaction of sulfur.To prevent particle growth, the reacted solution is quenched to freezingpoint of water and then stored in refrigerator.

Example I Aqueous synthesis of CdS QDs

1.6×10⁻⁴ mol Cd(NO₃)₂, 1.6×10⁻⁴ mol Na₂S, and 3.2×10⁻⁴ mol MPA,respectively, were prepared and each was dissolved into about 33 mldeionized water with stirring. The Cd(NO₃)₂ solution was added to theMPA solution at 2 ml/min with continuous stiffing. NH₄OH (1 M) was addedto the mixed solution to adjust the pH value to a pH of about 7-9. TheNa₂S solution was then quickly poured into the mixed solution andstirred for about 3-5 min. All of the above process steps were performedin an oxygen-free environment. In the present example, these steps wereperformed in a sealed glove bag pumped with nitrogen flow. Anultrasonicator was used to apply sonication for about 5-10 min. Largeagglomerates were removed by filtration, as necessary. CdS nanoparticleswere obtained in a clear suspension. The suspension was quenched in afreezer to 0° C., and the suspension was stored in a refrigerator atabout 4° C.

Example II Aqueous synthesis of ZnS QDs

The procedure was the same as in the example I, except that the chemicalprecursor Cd(NO₃)₂ was replaced by Zn(NO₃)₂. After the ZnS QDs wereprepared, the suspension was refluxed at 100° C. for several hours tohelp improve the photoluminescence properties.

Example III Aqueous Synthesis of CdS QDs with ZnS Doping

0.8×10⁻⁴ mol Cd(NO₃)₂ and 0.8×10⁻⁴ mol Zn(NO₃)₂, respectively, wereprepared and each dissolved into 16 ml deionized water with stiffing.1.6×10⁻⁴ mol Na₂S and 3.2×10⁻⁴ mol MPA, respectively, were weighed andeach dissolved into 33 ml deionized water with stirring. The Cd(NO₃)₂solution was added into the MPA solution and then the Zn(NO₃)₂ solutionwas added into MPA solution at about 2 ml/min with continuous stiffing.Thereafter, the process described in Example I was followed. The CdSwith ZnS doping (1:1) QDs are obtained as a clear suspension. Otherdoping ratios can be employed by adjusting the Cd to Zn molar ratio.

Quantum Yield

To obtain the quantum yield of the aqueous CdS QDs, Rhodamine 101 wasused as the standard sample, and the commercial QDs were compared. Allthe measurements were performed at the same conditions for Rhodamine101, for the commercial EviTags™ CdSe/ZnS QDs and for the aqueous CdSQDs of the present invention. For each sample, the absorbance andintegrated photoluminescence (PL) intensity were collected at a fixedexcitation wavelength 365 nm for several different concentrations. Theabsorbance was always kept below 0.15 to eliminate the re-absorptioninteraction effect. Using Rhodamine 101 dissolved in ethanol as thestandard sample with known quantum yield of 100%, the quantum yield ofEviTags™ CdSe/ZnS QDs was found to 8.9%, and the quantum yield of theaqueous CdS QDs was 6.0%. Without the core-shell structure, the aqueousCdS QDs have achieved high quantum yield comparable to that of thecommercially available EviTags™. FIG. 2 shows integratedphotoluminescence intensity vs. absorbance for Rhodamine 101, EviTags™CdSe/ZnS QDs and aqueous CdS QDs.

Concentration Effect

It was found that the PL properties of the aqueous QDs depend on theprecursor concentration. For the samples synthesized at different Cdprecursor concentrations of from 0.1 mM to 6.4 mM, the emission spectravaried. There was no linear relationship between the PL intensity of CdSQDs and the precursor concentration. FIG. 3 shows the normalized PLintensity of aqueous QDs for several precursor concentrations. Afternormalization based on the concentration, the QDs with the lowestprecursor concentration displayed the highest PL intensity. It isspeculated that at high precursor concentration, not all precursor wasnucleated and grown into particles. Perhaps the excess precursorsremained in the solution and didn't contribute to the PL intensity.

Stability

The CdS QDs suspension obtained using a precursor concentration of 1.6mM was mixed at a volume ratio of 1:3 with de-ionized water, phosphatebuffer solution (PBS (1×)) and cytosol (10⁶ cells/ml), respectively.FIG. 4 shows the PL intensity of aqueous QDs in the three solutions forup to 26 days, during which the samples were stored in the refrigeratorat 4° C. away from normal daylight. The samples were taken out of therefrigerator for PL measurements on the designated days. It was shownthat the aqueous CdS QDs have good stability with de-ionized water, PBSand cytosol. The results indicate that the aqueous QDs can be used asexcellent fluorescence markers in biological solutions for biomedicalimaging over a long period of time due to their stability.

Effects of ZnS Doping

ZnS was added to the CdS QDs to create different wavelengths ofphotoluminescence. FIG. 5 shows the PL intensity of ZnS-doped CdS QDs atseveral different ZnS contents. As the concentration of ZnS increases,the PL peak shifts toward smaller wavelengths. However, the PL intensityalso decreases with increasing amount of ZnS.

ZnS: Effect of Reflux

Pure ZnS QDs were synthesized as a candidate for nontoxic quantum dots.Zn is not harmful to the human body and thus ZnS would be suitable foran in vivo study. Refluxing the ZnS QD suspensions can increase the PLintensity as shown in FIG. 6. Refluxing for too long is detrimentalresulting in agglomerated QDs. For 1.6 mM ZnS QDs, refluxing for 16 hrsgave the best intensity.

Another alternative is to employ hydrothermal refluxing at hightemperatures.

It is to be understood that even though numerous characteristics andadvantages of the present invention have been set forth in the foregoingdescription, together with details of the structure and function of theinvention, the disclosure is illustrative only, and changes may be madein detail, especially in matters of shape, size and arrangement of partswithin the principles of the invention to the full extent indicated bythe broad general meaning of the terms in which the appended claims areexpressed.

References, disclosures of which are hereby incorporated by reference intheir entirety.

-   ¹Quantum Dots Get Wet, Science, volume 300, p. 80, Apr. 4, 2003.-   ²S. G. Penn, L. He, and M. J. Natan, “Nanoparticles for    Bioanalysis”, Curr. Opin. Chem. Bio., 7, 1-7, (2003)-   ³M. L. Brongersma, “Nanoshells, “Gifts in a Gold Wrapper”, Nature    Materials, vol. 2, May 2003.-   ⁴Applied Nanoworks. http://www.appliednanoworks.com/-   ⁵Evident Technologies. http://www.evidenttech.com.-   ⁶S. Foglia, L. Suber, and M. Righini, “Size Tailoring of CdS    Nanoparticles by Different Colloidal Chemical Techniques”, Colloid &    Surfaces, 177, 3-12, (2000)-   ⁷Z. Li and Y. Du, “Biomimic Synthesis of CdS Nanoparticles with    Enhances Luminescence”, Mater. Lett., 57, 2480-2484, (2003)-   ⁸R. Ko, C. L. Torres-Martinez, and R. K. Mehra, “A Simple Colloidal    Synthesis for Gram-Quantity Production of Water-Soluble ZnS    Nanocrystal Powders”, J. Colloid and Interf: Sci., 227, 561-566,    (2000)-   ⁹W. C. W. Chan, D. J. Maxwell, X. Gao, R. E Bailey, M. Han, and S.    Nie, “Luminescent Quantum Dots for Multiplexed Biological Detection    and Imaging,” Curr. Opin. Biotech., 13, 40-46 (2002)-   ¹⁰B. O. Dabbousi, J. Rodriguez-Viejo, F. V. Mikulec, J. R. Heine, H.    Mattoussi, R. Ober, K. F. Jensen, and M. G. Bawendi, “(CdSe)ZnS    Core-Shell Quantum Dots: Synthesis and Characterization of a Size    Series of Highly Luminescent Nanocrystallites,” J. Phys. Chem. B,    101, 9463-9475 (1997)-   ¹¹M. Bruchez, M. Moronne, P. Gin, S. Weiss, and A. P. Alivisatos,    “Semiconductor Nanocrystals as Fluorescent Biological Labels,”    Science 281, 2013-2015 (1998)-   ¹²D. Gerion, F. Pinaud, S. C. Willimas, W. J. Parak, D. Zanchet, S.    Weiss, and A. P. Alivisatos, “Synthesis and Properties of    Biocompatible Water-Soluble Silica-Coated CdSe/ZnS Semiconductor    Quantum Dots,” J Phys Chem B, 105, 8861-8871 (2001)-   ¹³M. Green, R. Taylor, and G. Wakefield, “The Synthesis of    Luminescent Adenosine Triphosphate Passivated Cadmium Sulfide    Nanoparticles,” J. Mater. Chem., 13, 1859-1861 (2003)-   ¹⁴A. L. Rogash, L. Katsikas, A. Kornowski, Dangsheng Su, A.    Eycmuller, and H. Weller, Synthesis and Characterization of    Thiol-Stabilized CdTe Nanocrystals”, Ber. Bunsenges. Phys. Chem.,    100, 1772-1778 (1978)-   ¹⁵B. V. Enustun and J. Turkevich, “Coagulation of Colloidal    Gold”, J. Am. Chem. Soc., 85, (21), 3317-3328, (1963)-   ¹⁶M. K. Chow and C. F. Zukoski, “Gold Sol Formation Mechanisms: Role    of Colloidal Stability”, J. Colloid & Interf: Sci., 165, 97-109,    (1994)-   ¹⁶L. E. Brus, J. Chem. Phys., 80, 4403 (1984)

1. A method for making quantum dots suitable for bioconjugation, saidmethod comprising the steps of: a. providing an aqueous solutioncomprising at least one water soluble metal salt suitable for forming aquantum dot core and at least one water soluble capping agent having areactive group capable of reacting with the quantum dot core and areactive group suitable for bioconjugation, and the solution has a pHaway from the isoelectric point of the quantum dot core; and b. addingat least one water soluble salt which is capable of reacting with atleast one metal of the at least one metal salt to form the quantum dotcore in order to form a suspension of quantum dots suitable forbioconjugation that do not have a non-excitonic broadband emissionbetween 500 nm and 700 nm.
 2. A method as claimed in claim 1, whereinthe broadband emission is at least 50 nm in width.
 3. A method asclaimed in claim 1, wherein the broadband emission is at least 100 nm inwidth.
 4. A method as claimed in claim 1, further comprising the stepof: c. quenching the quantum dot suspension to the freezing point ofwater.
 5. A method as claimed in claim 4, further comprising the stepof: d. conjugating the suspended quantum dots to a material directly inthe aqueous suspension, wherein the conjugated quantum dots are formedin a single aqueous process.
 6. A method as claimed in claim 1, whereinthe reactive group capable of reacting with the quantum dot core is athiol group.
 7. A method as claimed in claim 6, wherein the at least onecapping agent is selected from the group consisting of: mercaptosilanes,mercaptocarboxylic acids, and aminothiols.
 8. A method as claimed inclaim 7, wherein the at least one capping agent is selected from thegroup consisting of: 4-aminothiophenol,3-mercaptopropyltrimethoxysilane, mercaptoacetic acid, mercaptopropionicacid, mercaptosuccinic acid, mercaptobenzoic acid, andmercaptoundecanoic acid.
 9. A method as claimed in claim 1, wherein thequantum dot core comprises at least one metal sulfide selected from thegroup consisting of ZnS, PbS and MnS.
 10. A method as claimed in claim9, wherein the quantum dot core consists essentially of ZnS.
 11. Amethod as claimed in claim 1, wherein the quantum dot core comprises amixture of two or more metal sulfides.
 12. A method as claimed in claim1, wherein the at least one water soluble metal salt is selected fromthe group consisting of: Cd(NO₃)₂, Cd(ClO₄)₂, CdCl₂, CdSO₄, cadmiumacetate, Zn(NO₃)₂, Zn(ClO₄)₂, ZnSO₄, ZnCl₂, zinc acetate, Mn(NO₃)₂,Mn(ClO₄)₂, MnSO₄, MnCl₂, manganese acetate, Pb(NO₃)₂, Pb(ClO₄)₂, PbSO₄,PbCl₂, and lead acetate.
 13. A method as claimed in claim 6, wherein themolar ratio of thiol groups to total metal of the quantum dot is fromabout 1 to about
 5. 14. A method as claimed in claim 1, wherein themethod is performed in an atmosphere that is substantially free ofoxygen.
 15. A method for making quantum dots, said method comprising thesteps of: a. providing an aqueous solution comprising at least one watersoluble metal salt suitable for forming a quantum dot core and at leastone water soluble capping agent having a reactive group capable ofreacting with the quantum dot core and a reactive group suitable forbioconjugation, and the solution has a pH away from the isoelectricpoint of the quantum dot core; b. adding at least one water soluble saltwhich is capable of reacting with at least one metal of the at least onemetal salt to form the quantum dot core in order to form a suspension ofquantum dots suitable for bioconjugation; and c. quenching thesuspension of quantum dots to the freezing point of water.
 16. A methodas claimed in claim 15, further comprising the step of conjugating aprotein with the at least one capping agent of the quantum dot.
 17. Amethod as claimed in claim 16, wherein the protein is an antibody.
 18. Amethod as claimed in claim 17, wherein the at least one capping agentcomprises a thiol group and at least one group selected from a silanegroup and a carboxyl group.
 19. A method as claimed in claim 18, whereinthe at least one water soluble salt which is capable of reacting with atleast one metal of the at least one metal salt to form the quantum dotcore is a sulfide.
 20. Quantum dots suitable for bioconjugation made bythe process of claim 1.